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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
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Analysis of binding sites on complement factor I using artificial N-linked glycosylation

Jose I Sanchez-Gallego1, Tom W L Groeneveld, Stefanie Krentz

  • 1Department of Laboratory Medicine, Medical Protein Chemistry, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden.

The Journal of Biological Chemistry
|March 7, 2012
PubMed

Insights

Factor I (FI) regulates complement pathways by degrading C3b and C4b. Mutations in its FIMAC and SP domains significantly impair this function, highlighting their crucial role.

Area of Science:

  • Immunology
  • Proteomics
  • Biochemistry

Background:

  • Factor I (FI) is a serine protease essential for inhibiting complement pathways.
  • FI degrades activated complement components C3b and C4b, requiring cofactors like Factor H.
  • FI comprises a light chain (serine protease domain) and a heavy chain (FIMAC, CD5, LDLr1/2 domains).

Purpose of the Study:

  • To elucidate the functional importance of different Factor I domains in complement regulation.
  • To identify key protein-protein interaction sites within Factor I using homology modeling and mutagenesis.
  • To understand how mutations affect FI's cofactor-dependent degradation of C3b and C4b.

Main Methods:

  • Homology-based three-dimensional modeling of Factor I domains.
  • Site-directed mutagenesis to create 20 recombinant FI mutants with altered glycosylation sites.
  • Biochemical assays measuring FI's degradation of C3b and C4b in fluid and surface phases with various cofactors.

Main Results:

  • Mutations in the FIMAC and SP domains severely inhibited FI's ability to degrade C3b and C4b, regardless of the cofactor.
  • Alterations in the CD5 and LDLr1/2 domains had a less significant impact on FI's degradation activity.
  • The Michaelis constant (K(m)) for small substrates remained unchanged in all mutants.

Conclusions:

  • The FIMAC and SP domains are critical for Factor I's enzymatic activity in complement regulation.
  • Cofactors likely form similar complexes with FI and C3b/C4b, emphasizing the importance of domain accessibility.
  • Accessible FIMAC and SP domains are essential for the proper function of Factor I in inhibiting complement.

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